G protein-coupled receptors (GPCRs) are ubiquitously involved in regulation of vital cellular processes. In addition to ligand-induced activation, a small fraction of receptors may undergo a spontaneous conformational change, rendering ligand-independent activation. While ligand-independent, constitutive beta2-adrenoceptor (AR) signaling has been demonstrated in transgenic mice overexpressing cardiac beta2-AR, it is unclear whether the spontaneous activation is an intrinsic property of all GPCRs. In the present study, we intend to determine whether the dominant cardiac beta-AR subtype, beta1-AR, shares the ability of spontaneous activation. To exclude complications of endogenous ligands and native receptors, we created a pure beta1- or beta2-AR system via re-expressing beta-AR subtypes seperately in beta1- & beta2-AR double knockout (DKO) mouse myocytes using adenoviral (Adeno-beta1AR or -beta2AR) infection technique. To fulfill these goals, we first modified and improved current enzymatic methods to isolate high yield (0.53+/-0.55 million per left ventricle), high quality (83.8+/-2.5% rod-shaped) adult mouse cardiomyocytes. A practicle technique was also developed to culture these isolated myocytes and maintain their morphorlogical as well as physiological integrity after culture for 1-3 days. The high percentage of variable myocytes after 1 day culture (72.5+/-2.3%) allowed us to perform many biochemical assays, such as cAMP measurement, receptor radioligand binding, and Northern or Southern blot. Furthermore, the adenovirus- mediated gene expression is highly efficient in these cultured adult mouse myocytes.Re-expression of beta1- or beta2-AR after 1 day infection with recombinant adenovirus fully restored the contractile response to a mixed beta-AR agonist, isoproterenol, in DKO myocytes. Increasing the titer of Adeno-vectors (moi 10, 100 & 1000) led to a dose-dependent expression of beta1-or beta2-AR with maximal levels of 1425+/-288 and 821+/-38 fmol/mg protein, respectively. In the absence of ligand, graded increases in beta2-AR density resulted in corresponding increases in cAMP and contraction (with 428% and 233% increase, respectively, at a 68-fold increase in beta2-AR density). These effects were specifically reversed by a beta2-AR inverse agonist (which stabilizes the receptor in the inactive state), ICI118551 (5X10- 7 M). Thus, acute beta2-AR overexpression, similar to chronic beta2-AR overexpression in transgenic mice, increases spontaneous activation of the receptor and results in physiological responses. In contrast, overexpression of beta1-AR, even at a maximal density (46-fold), cannot enhance basal cAMP or contraction, suggesting that beta1-AR, unlike beta2-AR, does not bear agonist-independent, constitutive activation. We conclude that while beta1- and beta2-AR subtypes coexist in cardiac myocytes and share many similarities, beta1- and beta2-AR differ strikingly in their ability to undergo spontaneous activation. - beta- adrenergic receptor, cardiac myocyte, signal transduction, culture, gene transfer